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Expression and functional characterization of recombinant human HDAC1 and HDAC3

机译：重组人HDAC1和HDAC3的表达和功能表征

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Historic deacetylases (HDACs) are a family of enzymes involved in transcription regulation. HDACs are known to play key roles in the regulation of cell proliferation; consequently, inhibition of HDACs has become an interesting approach for anti-cancer therapy. However, expression of mammalian HDACs has proven to be difficult. All attempts to express these HDACs in E.coli, Pichia and baculovirus systems were unsuccessful. Here we present the stable expression of human recombinant His-tagged HDAC1 and HDAC3 in mammalian cells. Full-length human genes for HDAC1 and HDAC3 were cloned into the pcDNA 3.1 vector containing a N-terminal His-tag with an enterokinase cleavage site. Recombinant HDAC enzyme activity was only detected after nickel affinity purification due to high activity of endogenous HDACs; and removal of the His-tag increased activity 2-4 fold. Western blots demonstrated the nickel affinity purified rhHDAC1 preparation also contained endogenous HDAC2 and HDAC3; likewise, rhHDAC3 preparation contained endogenous HDAC1 and HDAC2. Therefore, the active HDAC preparation is actually a multi-protein and a multi-HDAC containing complex. This provides one explanation for the similar IC50 values exhibited by SARA and MS-275 against nuclear HDACs and rhHDAC1 and 3 preparations. These results demonstrate that recombinant forms of the HDACs can be over-expressed ill mammalian cells, isolated as active multi-protein complexes that contain multiple HDAC enzymes, and caution must be used when determining HDAC inhibitor in vitro selectivity. (C) 2004 Elsevier Inc. All rights reserved. [References: 34]
机译：历史性脱乙酰基酶（HDAC）是涉及转录调控的一族酶。众所周知，HDAC在细胞增殖的调控中起关键作用。因此，抑制HDAC已成为抗癌治疗的有趣方法。但是，已经证明哺乳动物HDAC的表达是困难的。在大肠杆菌，毕赤酵母和杆状病毒系统中表达这些HDAC的所有尝试均未成功。在这里，我们介绍人类重组组氨酸标签的HDAC1和HDAC3在哺乳动物细胞中的稳定表达。将HDAC1和HDAC3的全长人类基因克隆到pcDNA 3.1载体中，该载体包含一个带有肠激酶切割位点的N端His标签。由于内源性HDAC的高活性，仅在镍亲和纯化后才检测到重组HDAC酶的活性。和去除His标签增加了2-4倍的活性。 Western印迹证实，镍亲和纯化的rhHDAC1制剂还包含内源性HDAC2和HDAC3。同样，rhHDAC3制剂也包含内源性HDAC1和HDAC2。因此，活性HDAC制剂实际上是一种包含多种蛋白质和多种HDAC的复合物。这为SARA和MS-275对核HDAC和r​​hHDAC1及其3制剂表现出的相似的IC50值提供了一种解释。这些结果表明，HDACs的重组形式可以在哺乳动物细胞中过度表达，可以分离为含有多种HDAC酶的活性多蛋白复合物，在确定HDAC抑制剂的体外选择性时必须谨慎。 （C）2004 Elsevier Inc.保留所有权利。 [参考：34]

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